doi:10.1182/blood-2002-03-0896Prepublished online August 29, 2002;
and Paul W KincadeMaria Isabel D Rossi, Takafumi Yokota, Kay L Medina, Karla P Garrett, Philip C Comp, Arthur H Schipul developmental age related changesB lymphopoiesis is active throughout human life, but there are
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B lymphopoiesis is active throughout human life, but there are
developmental age related changes
(Running title: Age related changes in human B lymphopoiesis)
Maria Isabel D. Rossi*#, Takafumi Yokota*#, Kay L. Medina*, Karla P. Garrett*, Philip C.
Comp¶, Arthur H. Schipul, Jr.† and Paul W. Kincade*
*Immunobiology & Cancer Program, Oklahoma Medical Research Foundation
825 N.E. 13th Street, Oklahoma City, OK 73104
¶Department of Medicine, University of Oklahoma Health Sciences Center
Oklahoma City, OK 73104
†St. Anthony Hospital, Oklahoma City, OK 73101
#The first two authors contributed equally to this work.
Scientific heading: Immunobiology
This work was supported by grants AI 20069 and AI 45864 from the National Institutes
of Health.
Address correspondence to:
Paul W. Kincade, Immunobiology & Cancer Program, Oklahoma Medical Research
Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104
Tel: (405) 271-7905, fax: (405) 271- 8568, [email protected]
Word counts: Abstract 237 words / total text 5404 words
Copyright (c) 2002 American Society of Hematology
Blood First Edition Paper, prepublished online August 29, 2002; DOI 10.1182/blood-2002-03-0896 For personal use only. by guest on June 2, 2013. bloodjournal.hematologylibrary.orgFrom
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Abstract
This study addressed several questions concerning age related changes in
human B lymphopoiesis. The relative abundance of pro-B, pre-B, immature, naïve and
mature B cells among the CD19+ lymphocyte fraction of human bone marrow was found
not to change appreciably over the interval between 24 and 88 years of age. Moreover,
proliferation of pro-B and large pre-B cells in adult marrow equaled that observed with
fetal marrow specimens. Exceptionally low numbers of lymphocyte precursors were
found in some marrow samples and the values obtained were used to determine
parameters that best reflect B lymphopoiesis. Cord blood always contained higher
incidences of functional precursors than adult cells. However, sorted CD34+ Lin- CD10+
progenitors from cord blood and adult marrow had equivalent potential for differentiation
in culture and notable age related changes were found in more primitive subsets. A
recently described subset of CD34+ CD38- CD7+ cord blood cells had no exact
counterpart in adult marrow. That is, all adult CD34+ Lin- CD7+ CD10- cells expressed
CD38, displayed less CD45RA and had little B lineage differentiation potential. The
CD7+ fractions in either site contained progenitors for erythroid and NK lineages and
ones sorted from marrow expressed high levels of transcripts for the CD122 IL-2/IL-15
receptor required by NK lineage precursors. Dramatic changes in human B
lymphopoiesis occur early in life and more information is required to construct a
probable sequence of differentiation events prior to the acquisition of CD10.
Corresponding Author: [email protected]
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Introduction
Humoral immune responses are known to be compromised in the aged, and
decreased numbers of B and T lymphocytes parallel increases in NK cells.1-3 Animal
studies have revealed age related changes in B lymphocyte production within bone
marrow.4-6 Numbers of pro-B cells that can be resolved by flow cytometry are relatively
stable with age, but pre-B cells decline substantially.4,7 Incorporation of BrdU into
dividing cells indicates that B cell production is diminished in old mice.7 This
corresponds to the reduced entry of newly formed cells to the long-lived lymphocyte
pool.7,8 Functional precursor activity may decline because of a diminished support
capacity by marrow stromal cells.4,9 All of these observations indicate that peripheral B
lymphocyte populations in the aged may be replenished with fewer cells from marrow,
with potential consequences for the repertoire of available antibody specificities.
However, it is unclear if such findings are completely applicable to humans.
Animal studies have also been informative about molecular mechanisms and
steps in B lymphopoiesis. Rare hematopoietic stem cells transit a number of
“compartments” that are defined on the basis of cell surface markers. In reality, this
represents a continuum where genes are expressed and repressed in a temporal, but
not necessarily synchronous fashion. Early precursors have multiple differentiation
options and may express transcription factors in a way that does not reflect their
ultimate fate.10,11 Cells with potential for differentiation to lymphoid, but not myeloid or
erythroid cells have been referred to as common lymphoid progenitors.12,13 These Lin-c-
kitLoTdT+IL-7Rα+/Lo precursors likely derive from early lymphoid progenitors in the very
rare Lin-c-kitHiSca-1HiCD27+ fraction of bone marrow cells.14,15 However, it is important
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to note that the differentiation pathways utilized during fetal life may be quite different
from adult bone marrow.16-18
Knowledge of early B lymphocyte progenitors in humans lags behind that for
mice, and there are important differences. IL-7 is essential to this process in mice but
not in humans, and no combination of cytokines has been identified that supports
vigorous human lymphocyte formation in culture.19-21 Indeed the most effective models
for human B lymphopoiesis use murine stromal cells that provide uncharacterized
stimuli.22 Cytokines and hormones have been identified that selectively inhibit B
lymphocyte formation in mice.13,23-27 It is important to know if lymphopoiesis is altered
as humans age because such negative factors increase. Some investigative
techniques are not appropriate with specimens of human marrow, and the yield of
hematopoietic cells is variable. Cellularity decreases with age in some but not all
human bones,28,29 and most studies have been performed on marrow aspirates
contaminated by peripheral blood.30,31
Despite these species differences and technical difficulties, progress is being
made in understanding the process in humans. For example, there is evidence that
CD10+CD19- cells may be the human equivalent of common lymphoid progenitors in the
mouse, while candidates recently identified in umbilical cord blood are CD34+CD38-
CD7+.32,33 A major goal of this study was to determine if numbers and functions of such
precursors decline with age, because that could reflect important changes in human
lymphopoietic capacity.
Expression of Rag genes in marrow of mature individuals might suggest that Ig
genes are being rearranged.34,35 Unfortunately, little information has been available
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about proliferation36,37 or differentiation activity in humans, and it is possible this slows
with age. Thus, while the incidence of lymphocytes has been known to decline between
birth and puberty, there was an insufficient understanding of changes that may occur
subsequently.31,35,38
Ratios of precursor subsets in human marrow, proliferative indices, and
differentiation were evaluated in the present study with the goal of identifying age
related changes. We now report that while B cell precursors are depressed in some
patients, the entire differentiation series is normally present throughout life and is
mitotically active. However, surface marker changes occur early in life, and adult
marrow has reduced potential for lymphocyte production in two model systems.
Additional information about acquisition of CD7 and CD10 may prove helpful in
understanding the sequence of early differentiation events in humans.
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Materials and Methods
Cell Sources. Cord blood samples were obtained from placentas of healthy newborns
collected at the Oklahoma University or Saint Anthony Hospitals (Oklahoma City, OK).
Adult marrow samples were collected from patients between 24- to 88-years of age that
were undergoing hip replacement surgery at the Bone and Joint Hospital (Oklahoma
City, OK). Patient records with no identifying information were used to determine that
some individuals had a recent history of treatment with anti-inflammatory drugs such as
prednisone or the drug Naprosyn. Most specimens came from otherwise healthy
patients with no evidence of malignancy or immunosuppression. The Anatomic Gift
Foundation (Laurel, MD) supplied femoral bone marrow from fetuses between 16 and
24 weeks of gestation. An Investigational Review Board approved all procedures
involving human specimens.
Animals. NOD/LtSz-scid/scid (NOD/SCID) mice were obtained from a breeding colony
established at the Oklahoma Medical Research Foundation (OMRF, Oklahoma City,
OK) from breeding pairs kindly provided by Dr. Leonard D. Schultz (The Jackson
Laboratory, Bar Harbor, ME). Animals were housed in a restricted barrier facility under
specific pathogen-free conditions.
Antibodies and Cytokines. The following antibodies were either purified, biotinylated or
conjugated with FITC, phycoerythrin (PE) or allophycocyanin (APC): anti-CD38 (clone
HIT2), anti-CD24 (clone ML5), anti-CD23 (clone M-L233), anti-CD45 (clone HI30), anti-
Ki-67 (clone B56), anti-IgD (clone IA6-2) from PharMingen, (Becton & Dickinson, San
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Jose, CA), anti-CD34 (HPCA-2) from BD (Becton & Dickinson), anti-CD3 (clone
UCHT1), anti-CD7 (clone 6B7), anti-CD8 (clone HIT8a), anti-CD14 (clone TUK4), anti-
CD16 (clone 3G8), anti-CD10 (clone 5-1B4), anti-CD19 (clone SJ25-C1), anti-CD13
(clone TUK1), anti-CD27 (clone CLB-27/1), anti-CD33 (clone 4D3), anti-CD56 (clone
NKI nbl-1), anti-CD64 (clone 10.1), anti-κ light chain (clone HP6062), anti-λ light chain
(clone HP6054), and anti-glycophorin A (clone CLB-ery1) from Caltag Lab. (Burlingame,
CA). F’(ab) fragments of goat anti-human IgM or anti-human IgD were from Southern
Biotechnology Associates Inc. (Birmingham, AL). The biotinylated antibodies were
revealed with Streptavidin Red 613 (Gibco, BRL, Rockville, MA). Purified
rherythropoietin (EPO), rhSCF, rhFlt3 ligand, rhIL-15 and rhG-CSF were purchased
from R & D Systems (Minneapolis, MN).
Cell isolation and staining procedure for FACS analysis. Four- color
immunofluorescence analysis was used for the identification of the different B cell
precursor populations in total nucleated bone marrow cell suspensions from fetal and
adult marrow. Briefly, single cell suspensions were obtained by flushing or gently
vortexing the bone marrow. After staining, erythrocytes were lysed using the B-D lysis
buffer solution according to the manufacturer’s instructions and CD45 antibody was
used to assess the percentages of leukocytes in each sample. Intracellular staining
was done by permeabilizing cells after surface staining. The cells were fixed with 1%
paraformaldehyde in PBS and permeabilized with 70% ethanol at –20ºC, for more than
30 minutes and washed twice with the staining buffer. They were then incubated with
anti-human IgM for 30 min at room temperature. To establish the proliferative fraction,
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cells were incubated with the antibody anti-Ki67 (mib-1) for 1 hour on ice, as described
by Zupo et al.39 Data acquisition and analyses were performed on a FACSCalibur
(Becton Dickinson).
Isolation of CD34+ cells and cell sorting. Mononuclear cells were collected from cord
blood or adult marrow by Ficoll/Hypaque (Lymphocyte Separation Medium, Cellgro-
Mediatech, Herndon, VA) centrifugation. Enrichment for CD34+ cells was performed
using the Direct CD34 Isolation Kit (Miltenyi Biotechnology (Auburn, CA). CD34+
enriched cells were stained with CD34-FITC, lineage markers (Lin, CD13, CD14, CD33
and CD64 for myeloid cells, glycophorin A for erythroid cells, CD19 for B cells, CD3 and
CD8 for T cells, CD56 for NK cells)-PE, CD7-biotin followed by Streptavidin Red613,
and CD10-APC. CD34+Lin-CD10-CD7-, CD34+Lin-CD10-CD7+, and CD34+Lin-
CD10+CD7- cells were sorted using a MoFlo (Cytomation, Fort Collins, CO). The sorted
cells were subsequently subjected to flow cytometry, culture, and gene expression
analyses. In some experiments, CD34+CD38- cells were sorted and cultured. In this
case, mononuclear cells were incubated with mouse anti-human antibodies: CD3,
CD13, CD14, CD16, CD33, CD56 and glycophorin A, and negatively selected using the
BioMag goat anti-mouse IgG-coated beads (PerSeptive Biosystems Inc., Framingham,
MA). The enriched cells were stained with CD34-FITC and CD38-PE. CD34+CD38-
cells were determined using gating previously described.40 Post-sort analysis revealed
the purity of the sort populations to be greater than 95%.
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Transplantation of human cells into NOD/SCID mice. Magnetically enriched CD34+
cells from cord blood, fetal or adult marrow were diluted in PBS with 0.1% BSA (bovine
serum albumin fraction V, Sigma Chemical Co.) and injected into the tail vein of sub-
lethally irradiated (100 cGy from a 137Cs source) 8- to 12-weeks old male or female
NOD/SCID mice. 5 x 106 CD34- cord blood mononuclear cells were co-injected as a
source of accessory cells.41,42 No chimerism was ever obtained in mice transplanted
with the accessory cells alone.
Limiting dilution assays (LDA). The frequencies of B cell progenitors in cord blood and
adult marrow were determined by plating CD34+CD38- cells in limiting dilution assays.
Between 20 and 180 wells containing pre-established MS-5 stromal cell layers (a kind
gift of Dr. J. Mori, Niigata University) were plated with 3, 5, 10, 15, 20, 30, 50, 75, 100,
150, or 200 cells each using the Automated Cell Deposition Unit of the MoFlo. Cells
were cultured in α-MEM (Cellgro-Mediatech) supplemented with 10% FCS (Hyclone,
Logan, UT) and a combination of rhSCF (100 ng/ml) and rhG-CSF (10 ng/ml).21 Wells
were inspected every week for the presence of new clones. Positive wells were
harvested after 5 to 7 weeks of culture, and analyzed by flow cytometry for CD19+ cells.
The frequencies of precursors were calculated by linear regression analysis on the
basis of Poisson distribution as the reciprocal of the concentration of test cells that gave
37% negative cultures.
Multil-lineage co-culture system and methycellulose culture. Sorted CD34+Lin-CD10-
CD7-, CD34+Lin-CD10-CD7+, and CD34+Lin-CD10+CD7- cells were cultured at 1 x 104
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cells per 6 mL/25-cm2 tissue culture flask (Corning, NY) with MS-5 stromal cells.
Culture media was α-MEM containing 10% FCS, rhSCF (100 ng/ml), rhG-CSF (10
ng/ml), rhFlt-3 ligand (100 ng/ml), and rhIL-15 (10 ng/ml). The cultures were maintained
with weekly whole medium changes and the generation of myeloid, B and NK cells was
evaluated at the indicated periods. Methylcellulose cultures were performed in 35mm
plates (Corning) using MethoCultTM GF H4534 medium (StemCell Technologies Inc.,
Vancouver, Canada) containing rhSCF, rhGM- CSF and rhIL3 along with 3 units/ml of
rhEPO (R&D Systems). All cultures were maintained at 37ºC in a humidified incubator
with 5% CO2 in air. Differential colony counts were scored after 10-14 days by
morphological characteristics using an inverted microscope and confirmed by staining
individual colonies with Wright’s or May-Grunwald-Giemsa stain.
Reverse transcriptase polymerase chain reaction (RT-PCR). Sorted cells were put in
lysis buffer (Ambion, Austin, TX) and mRNA was extracted using Poly-A column
(Ambion) according to the manufacturer’s instructions. cDNA was prepared from Dnase
I-treated mRNA using oligo-dT and Moloney murine leukemia virus reverse
transcriptase (Gibco BRL). Semiquantitaive PCR was done in order to measure relative
differences in transcript levels of target cDNAs against levels of the reference gene
GAPDH. The PCR reation was done in 100 µl containing 1 X PCR buffer (TaKaRa
Biomedical, Osaka, Japan), 1.5mM MgCl2, 200 µM dATP, dGTP, dTTP, 100 µM dCTP,
and 50 pmoles of each primer. For quantification, 0.5 µCi [alpha32P] dCTP (Amersham,
Arlington Heights, IL) was included in each reaction tube. Samples were denatured in a
DNA thermocycler (Perkin-Elmer, Norwalk, CT) for 10 min at 95ºC. To increase
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specificity, 2.5 U of Taq DNA polymerase (TaKaRa) was added to each sample during
this initial denaturation. Samples were then cycled for 1 min at 94ºC, 1 min annealing
60°C,1.5 min extension at 72ºC with a final extension of 7 min at 72ºC. Aliquots were
removed at cycles 25, 28, 31 and 34 for GAPDH and cycles 32, 35 and 38 for all others
to insure that PCR remained within the exponential range of amplification. 5 µl aliquots
were denaturated in a formamide loading buffer and applied to a 6% polyacrylamide gel
containing 7 M urea. Incorporation of [alpha32P] dCTP into PCR product bands was
quantified from dry gels using a PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
Primers were as follows: GAPDH-sense: 5’-TCCAAAATCAAGTGGGGCGAT-3’,
GAPDH-antisense:5’-TTCTAGACGGCAGGTCAGGTC-3’, 475- bp expected product,
RAG-sense: 5’-CCTGAGTCCTCTCATTGCTGAGAG-3’, RAG1-antisense: 5’-
AGGGCATGATGATCGCCATACT-3’, 681bp product , RAG-2-sense: 5’-
CTAATGAAGAGCAGACAACATTCA-3’, RAG2-antisense: 5’-
TAGGACTCTTTGGGGAGTGTGTAG-3’, 422bp product, EBF-sense: 5’-
CCGGGCTCACTTTGAGAAGCAG-3’, EBF-antisense: 5’-
CAGGGAGTAGCATGTTCCAGAT-3’, 638bp product, PAX-5-sense: 5’-
CTCGGTGAGCACGGATTCGGCC-3’, PAX-5-antisense: 5’-
GCGGCAGCGCTATAATAGTAG-3’, 621bp product, CD122-sense: 5’-
GCGTGGCTCGGCCACCTC-3’, CD122-antisense: 5’-
GACGATGAGGGGAAGGGCGAAGA-3’ ,211bp product, Id-1 as described.43
Statistical analysis. All results are shown as mean values ± S.D. Differences between
groups were assessed using the Student t test.
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Results
B lymphopoiesis persists throughout adult human life. It has been reported that
absolute numbers of B lymphocyte precursors in human marrow decline with normal
age, and particularly during adolescence.38 Therefore, we calculated percentages of
CD19+sIg- lymphocytes and CD19+sIg+ B cells relative to total numbers of nucleated
cells in our adult marrow samples (data not shown). As in the previous study,38 sample-
to-sample variations were considerable, but we saw no substantial or consistent
depletion of these lymphocyte populations as a function of age in adults. We then
extended previous studies31,35 describing B lineage lymphocyte precursors in adult
human marrow (Figure 1A). Pro-B cells (CD34+CD10+CD19+), pre-B cells (CD34-
CD19+sIg- or CD19+cµ+ sIg-) and immature B cells (sIgM+CD24hiIgD-CD10+CD38+)
represented relatively constant percentages of the CD19+ lymphocytes over a 24-88
year age span. The ratios between these populations and the presence of naïve (sIgMhi
IgD+CD24hiCD10+/-CD38+/-CD27-) B cells would all be consistent with stable B
lymphopoiesis. Mature recirculating B cells (sIgM+IgD+CD24loCD38- ) were previously
shown to comprise a large fraction of marrow lymphocytes,35. We found no mature B
cells in fetal bones, but there was no substantial influence of adult age on this
population (Figure 1A). Mature B cells in marrow were also CD27+, a characteristic of
somatically mutated memory B cells in peripheral blood.44 Most female marrow donors
were receiving estrogen replacement therapy and we found no sex related differences
in this analysis.
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Blood cell production results from massive division within marrow, and we
assessed expression of the Ki-67 nuclear proliferation antigen in B lineage lymphocyte
precursors (Figure 1B). Again, we found no age related changes in this parameter, and
male versus female values were comparable. A similar analysis was recently
performed with fetal marrow specimens,45 and the results are included here for
comparison (Figure 1B). With the exception of small pre-B cells, that are more
mitotically active during embryonic life, B cell precursors were remarkably similar with
respect to Ki-67 expression. We conclude that actively proliferating precursors are
present within the lymphocyte fraction of human marrow until at least the eighth decade.
Analysis of marrow from exceptional patients reveals useful indices of B cell production.
The data shown above would be consistent with stable B lymphopoiesis occurring over
a large age range. However, it is difficult to know the degree to which lymphopoietic
marrow is replaced by fat as a function of age and to control for sample-to-sample
variations with respect to peripheral blood contamination and location. Therefore, it
would be helpful to have other parameters for assessing marrow activity. Relatively
constant proportions of B lineage lymphocytes were found in most of the specimens we
examined (Figure 1A above). Whenever immature (IgM+CD24hiIgD-CD10+CD38+) B
cells were present, marrow samples contained mitotically active pre-B (CD19+sIgM-)
cells identified with the Ki-67 antibody. However, exceptionally low numbers were found
in samples obtained from some patients. There were insufficient numbers of these
individuals and information about specific treatments to formally assign them to groups.
However, the degree of sample-to-sample variation in these specimens provided an
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opportunity to seek correlations (Figure 2). In these individuals, incidences of cycling
pre-B cells predicted the presence of immature lymphocytes (p = 0.0001), consistent
with a close precursor-product relationship between the two. Naïve (IgM+CD24hiIgD+
CD38+CD10+/- D27-) B cells did not closely correlate with proliferating pre-B cells (p =
0.1035), although they are the progeny of immature B cells. We emphasize the
usefulness of Ki-67 expression, because simple percentages of pre-B cells did not
correspond as closely with numbers of newly formed lymphocytes (data not shown).
The findings suggest that the size of the proliferating pre-B cell pool determines
numbers of newly formed B cells.
Early developmental age related changes in populations resolved by flow cytometry.
Earlier reports demonstrated that CD7 could be used to discriminate hematopoietic
stem cells from more differentiated precursors of NK and T lymphocytes.46,47 It is also
noteworthy that CD7 is rarely co-expressed with CD10 or CD19, and there is evidence
that CD7+ cells in cord blood represent common lymphoid progenitors.33 However,
comparisons made by flow cytometry suggested that cord blood might not be
representative of differentiation pathways utilized in adult marrow. Adult CD34+ Lin-
cells contained a more conspicuous subset of CD7-CD10+ progenitors as well as more
CD7+CD10- cells (Figure 3A). Additional differences were found by comparing sorted
CD7+ and CD7- subsets of CD34+Lin-CD10- cells from the two sources (Figure 3B and
3C). It has been suggested that CD45RA is acquired before CD1032 and we found that
nearly half of the cord blood CD7+ cells expressed moderate to high levels (Figure 3B).
In contrast, only 10% of the comparable subset from adult marrow was CD45RA+. As
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noted above, the CD38- subset of the CD34+ Lin- CD7+ fraction of cord blood has been
recently found to contain multi-lymphoid, but not myelo-erythroid progenitors.33 It is
remarkable that cells with that combination of markers do not exist in adult marrow
(Figure 3C). Note that the CD7- subsets of fetal and adult populations were similar with
respect to CD45RA and CD38 expression. Thus, the composition of hematopoietic cell
subsets and/or surface marker display in cord blood differs substantially from adult
marrow.
Transplantation experiments reveal age related changes in differentiation potential.
Immunodeficient NOD/SCID mice were prepared for transplantation with low dose
irradiation (see Materials & Methods) and then injected with highly enriched
suspensions of CD34+ cells (Table 1). Previous studies documented an extraordinary
differentiation potential of cord blood stem cells in this model,48-50 so we compared one
log less CD34+ cells from this source to ones isolated from fetal and adult marrow. The
latter represented pools of CD34+ cells obtained from specimens ranging from 35 to 82
years of age in one experiment and 62 to 77 years of age in a second transplant. Flow
cytometry was performed on aliquots of the suspensions to determine numbers of
CD34+ CD38- cells injected because of reports that these cells are particularly effective
for engraftment of NOD/SCID mice51 (Table 1). It has been shown that undefined
accessory cells can influence engraftment of human cells in mice.41,42 Therefore, we
added 5 x 106 CD34- cord blood mononuclear cells to all samples of CD34+ cells before
transplantation and the recipients were assessed 7 weeks later. No human cells were
detectable in animals that received only the CD34- cells, but easily discernible
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populations of CD45+CD19+ lymphocytes were found in virtually all recipients of CD34+
cells, regardless of donor age (Table 1). Adult precursors were less effective than those
obtained from the other two sources with respect to numbers of lymphocytes produced,
suggesting a possible consequence of the early developmental age related changes
noted above.
Culture experiments reveal age-related differences in expansion and differentiation
potential of lymphocyte progenitors. Homing to the marrow, resistance to
xenotransplantation barriers and many other factors could influence the degree of
chimerism observed in the NOD/SCID model. Furthermore, culture experiments could
permit detailed analysis of small cell numbers. Therefore, we performed a limiting
dilution assay where graded numbers of CD34+CD38- cells were seeded onto
monolayers of murine MS-5 stromal cells and stimulated with SCF and G-CSF (Table
2). The frequency of precursors with the potential for clonal expansion varied from
3.6% to 9.78% in marrow suspensions obtained from normal healthy 24-88 year old
donors. In separate experiments, the cloning efficiency of umbilical cord blood CD34+
CD38- cells ranged from 11.11% to 14.29% using the same culture conditions. In this
case, the B cell progenitor frequency was 6.67% (5.56% to 8.33%). Furthermore, in
about 40% of the wells more than 50% of the cells produced by cord blood CD34+CD38-
cells were CD19+, and this was never the case with adult marrow. Analysis of pooled
clones revealed that differentiation did not proceed efficiently beyond the CD19+ stage
in such cultures and very few cytoplasmic or surface µ+ cells were recovered.
Culture conditions were then developed that allowed simultaneous differentiation
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of CD19+ B lineage, CD13/CD33+ myeloid lineage, and CD56+ NK lineage cells (see
Materials and Methods). Absolute numbers of CD19+ cells produced were equal to, or
more than those obtained with other methods we have tried and NK lineage cells were
always efficiently produced when IL-15 was present. The method was used for a side-
by-side comparison of fetal/neonatal versus adult marrow precursors. Three-week
cultures initiated with 104 CD34+Lin-CD7-CD10- cord blood precursors yielded much
larger numbers of B lineage lymphocytes than those prepared with equivalent cells from
adult marrow (Figure 4A). Interestingly, the lymphoid potential of CD34+Lin-CD7-
CD10+ cells from the sources was comparable. The CD34+Lin-CD7+CD10- subset was
efficient only when derived from cord blood. All subsets of cord blood yielded
approximately 10 times the number of CD56+ NK lineage cells than obtained in cultures
of their adult counterparts.
Myeloid potential was highest in the CD34+Lin-CD7-CD10- fractions, and
dramatically down regulated in CD7+ or CD10+ subsets, regardless of source. These
results are consistent with clonal assays conducted with methylcellulose cultures. With
two analyses of cord blood, most non-lymphoid progenitors were present in the CD7-
subset (710.8 ± 74.6 colonies / 1000 cells). However, it is noteworthy that an average
of 170.8 (± 50.8) colonies per 1000 cells were also obtained when the CD7+ fraction was
plated and 95% of them were erythroid. Similarly, the CD34+ Lin- CD7-CD10- fraction of
adult marrow contained most (an average of 60% in three experiments) of the
granulocyte/macrophage progenitors (CFU-G/M). As with cord blood cells, 98% of the
colonies obtained with the CD34+Lin-CD7+ fraction were erythroid (BFU-e). Insufficient
numbers of the rare CD7+CD10+ subset of adult marrow precluded their analysis in
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these cultures.
Weekly examination revealed that myeloid progeny appeared quickly and then
declined in cultures initiated with the CD34+Lin-CD7-CD10- fraction of cord blood, while
myelopoiesis was more persistent in cultures of their adult counterparts (Figure 4B, C).
Progressive production of B and NK lineage cells was consistent with the primitive
nature of progenitors in this fraction. While transient B lineage differentiation was found
with the CD34+Lin-CD7+CD10- fraction, this was never the case with the comparable
adult marrow subset. The adult population also yielded a wave of NK lineage cells as
contrasted with sustained production from neonatal cells.
Neonatal or adult CD10+ fractions contained B lineage precursors that
differentiated within two weeks in culture and CD56+ NK lineage cells reached peak
values one week later. This analysis revealed large age related changes in the
potential of highly purified cord blood and adult marrow progenitors to expand in culture.
CD10 and CD7 expression denote commitment to B and NK lineages. These findings
highlight the importance of learning more about the B lineage precursors in human
marrow. Therefore, adult subsets sorted for use in the culture experiments described
above were also subjected to RT-PCR analysis (Figure 5). Transcripts associated with
B lymphocyte lineage differentiation (RAG-1, RAG-2, EBF and Pax5) were markedly up
regulated in the CD34+Lin-CD10+CD7- population relative to the more primitive CD34+
Lin-CD10-CD7- subset. Furthermore, levels of the Id-1 transcriptional repressor were
reduced in that fraction (data not shown). In contrast, expression of the CD122 receptor
for IL-15/IL-2 required by NK lineage cells was strongly associated with display of CD7.
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Although transcripts for RAG1 and RAG2 were detectable in CD7+ cells, this fraction
was deficient in the EBF transcription factor required for B lymphopoiesis. Very similar
results were obtained in three independent experiments. These results complement
those obtained with culture experiments and suggest that acquisition of CD10 or CD7
corresponds to substantial progression in the B or NK lineages, respectively.
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Discussion
This study was informative about several aspects of human B lymphopoiesis.
Ratios between precursors and immature B cells within marrow, and an indication of
mitotic activity were found to be remarkably constant throughout adult life. Exceptional
marrow samples from some patients were deficient in lymphocyte precursors, providing
a unique opportunity to see how the abundance of particular subsets corresponds to
newly formed B cells. While the potential for lymphoid differentiation, as assessed by
culture and transplantation assays was retained in most individuals, the lymphocyte
yield from adult progenitors was much less than that obtained from cord blood.
Furthermore, we documented age related changes in subsets resolved on the basis of
CD7 and CD10 expression. Analysis of highly purified progenitors revealed that
erythroid and NK differentiation potential might diverge from B lineage potential as cells
acquire one or the other of these markers.
There are many technical problems associated with analysis of human marrow
specimens, and it is difficult to estimate absolute numbers of B cell precursors. As one
example, mature re-circulating B cells have been identified that have a distinctive
density of CD24 and sIgM.35 Absolute numbers of these cells per unit marrow or body
weight are not known, but they progressively dilute B cell precursors and newly formed
B cells. However, it is fortunate that other measurements reflect the lymphopoietic
activity in that organ. As in previous studies31,35 the ratios between B cell precursors
and newly formed B cells can be determined, and we confirm that they are relatively
constant. Moreover, the proliferative activity of B cell precursors was surprisingly stable
with age. In a separate study, we evaluated precursors in fetal marrow and found that
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an exceptionally large fraction of small pre-B cells in that site are Ki-67+.45 As discussed
below, this is one of several distinctive features of fetal lymphopoiesis. Once the
process is established in adult marrow, the proliferation index of B cell precursors
remains unchanged until at least 88 years of age.
The striking deficiency of B cell precursors in some of the marrow specimens
suggests that influences of therapy and environmental stress on B lymphopoiesis merit
further study. One previous report recorded delayed recovery of B cells after marrow
transplantation in patients receiving cortisone.52 Most of our deficient samples came
from patients who were being treated with anti-inflammatory drugs and we will show
elsewhere that human lymphocyte progenitors are very sensitive to glucocorticoids.
Here we utilized the variation provided by these unusual specimens to make inferences
about precursor – product relationships within marrow. The best correlation was found
between cycling pre-B cells and immature B cells. The Ki-67 proliferation antigen
provided a particularly useful index, and simple pre-B cell percentages did not correlate
as well. Whenever percentages of proliferating pre-B cells were beneath 50%,
immature (IgM+IgD-CD24hiCD10+CD38+) B cells were scarce. The correlation between
percentages of naïve CD38+ B cells and cycling pre-B cells was not as good as the
relationship between pre-B cells and immature B cells (Figure 2). Cells with a naïve
phenotype might randomly leave marrow to enter the peripheral B cell pool, as has
been suggested for newly formed B cells in mice.53
It was important to learn if cells with surface characteristics of B cell precursors
actually had the potential to give rise to lymphocytes. Human CD34+CD38- cells have
been determined in other studies to be responsible for engraftment of NOD/SCID mice,
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but uncharacterized accessory cells among the CD34- fraction of hematopoietic cell
suspensions can influence the outcome.41,42,51 To minimize this parameter, we added a
constant number of cord blood CD34- mononuclear cells to each of the transplant
samples, and human CD19+ cells were easily detectable in marrow of all transplanted
mice. As others have shown, stem cells within cord blood are exceptionally efficient for
engraftment of immunodeficient mice.48-50 We estimate that seven weeks after
transplantation there were 111 ± 98 human CD19+ cells present in each mouse femur
per injected CD34+CD38- cord blood cell. This value is 10 to 100 fold higher than we
obtained with precursors isolated from adult marrow.
Limiting dilution cultures initiated with highly purified CD34+CD38- cells provided
an independent means of assessing differentiation potential, and one that should be
less influenced by contaminating accessory cells. Cord blood precursors had a higher
cloning efficiency in vitro than adult marrow derived cells, and more of the responding
wells generated CD19+ lymphocytes (Table 2). This result is compatible with
observations made in another study, using a different culture system54 and suggests the
possibility of an intrinsic difference between neonatal versus adult progenitors.
Since the proportion of hematopoietic marrow relative to fat progressively
declines with age in long bones, total numbers of newly formed B cells might be
reduced in some locations.28 However, the cellularity of marrow in other sites is
remarkably constant throughout life, and it will be important to determine the total body
output of new B cells.29 This important issue awaits other types of analyses comparable
to those recently used to assess thymic activity in adult humans.55-57 Many studies
suggest that hematopoietic stem cells retain the potential for replenishing lymphocytes
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throughout life.58,59 However, there are changes with respect to incidence, homing
potential and function on a per cell basis. Additionally, there can be age-related
declines in the ability of environmental cells within marrow to support lymphopoiesis.4
Genetic polymorphisms influence stem cell numbers and senescence to a substantial
degree.59,60 For that reason, our culture assays were conducted with cells from
individual marrow donors. While flow cytometry revealed proportions of lymphocyte
precursors in most marrow specimens to be remarkably constant, functional precursor
frequencies among CD34+CD38- cells were highly variable (Table 2).
Flow cytometry experiments revealed that the composition and patterns of
surface marker expression differ substantially between cord blood and adult marrow.
The interesting CD34+CD38-CD7+CD10- subset recently described in cord blood33 has
no direct counterpart in adults. Furthermore, the cord blood CD34+CD38-CD7-CD10-
subset differed substantially from the comparable adult population with respect to
expression of CD45RA. Given these changes, we elected to sort three populations for
side-by-side comparisons in culture studies. CD10+CD19- cells comprise a very small
proportion (2.5±1.1%) of the 1% of marrow mononuclear cells that express CD34+ (30,32
and Figure 3). Most of these CD34+CD10+CD19- cells also express TdT30,32,61 and data
not shown) and already have D-JH rearrangements.34 Previous reports32,34 suggest that
this fraction is a rich source of early B lineage precursors. Our culture analyses
substantiate that conclusion and it is noteworthy that cord blood and adult progenitors
were equivalent in potential for B lineage differentiation (Figure 4). PCR analysis of
CD34+Lin-CD7-CD10+ cells from adult marrow revealed substantial expression of five
genes required for B lymphopoiesis (Figure 5). However, it is also important to stress
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that CD10+ cells have some myeloid potential that can be appreciated in long term
cultures but not in clonal assays32 (Figure 4 and data not shown). Thus, a completely
lymphoid restricted progenitor has not been isolated from adult human marrow.
The question arises whether B lineage associated events begin prior to the
CD10+ stage and CD7+ cells are interesting in this regard. One recent study found that
a CD34+CD38-CD7+ fraction of cord blood contains what might be the human equivalent
of common lymphoid progenitors described in mice.12,33 We confirm that cord blood
CD34+CD7+CD10-CD19- cells can rapidly generate small numbers of CD19+ cells in
culture (Figure 4 and data not shown). Unlike the previous report, we detected some
non-lymphoid, and especially erythroid precursors in this population. This may be
because we did not additionally select for CD38- cells in cord blood. However, as noted
above, there are no CD38-CD7+ cells in adult human marrow and the total CD34+Lin-
CD7+CD10- fraction of marrow was a poor source of B lineage progenitors (Figures 3
and 4). PCR analysis of carefully sorted cells substantiated the culture results and
showed a considerable enrichment for cells expressing the CD122 receptor for IL-15/IL-
2 required for NK lineage differentiation (Figure 5). It was previously reported that the
CD34+CD7+ fraction of adult marrow contains precursors of NK cells.46 Thus, CD7
expression may characterize an intermediate stage between stem cells, erythroid cells
and pro-B cells in neonates, but be more closely associated with NK lineage restricted
precursors in adult marrow.
To summarize, several distinctive adult characteristics of B lymphocyte
precursors may be acquired during the neonatal or adolescent period. The primitive
CD34+Lin-CD10-CD7- fraction retains differentiation potential throughout life, but is much
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less efficient than cord blood cells with the same phenotype. Cells bearing CD10+
include many early B lineage precursors and ones in neonates and adults appear to
have equivalent differentiation potential. All subsequent stages of B lineage
differentiation are represented in constant ratios in marrow and numbers of proliferating
pre-B cells closely correlate with numbers of newly formed B cells. The search must
continue for lymphoid restricted progenitors in human marrow and our understanding of
early differentiation events is incomplete.
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Acknowledgments
P.W.K. holds the William H. and Rita Bell Chair in biomedical research.
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Table 1. Adult human bone marrow contains B lymphocyte precursors revealed
by transplantation of immunodeficient NOD/SCID mice.
Cells InjectedNo. of cells
Injected
No. of
CD34+CD38-
cells injecteda
No. of
engrafted/injected
mice (%)
Percentage of
hCD45+CD19+
cells in BM of
chimeras
Fetal BM CD34+ 2.9 – 3 X 106 28.5 – 30 x 103 5/6 (83%) 1.54 (± 1.28)
CB CD34+ 5 X 105 4 x 103 11/11 (100%) 1.02 (± 0.86)
Adult BM CD34+ 2 X 106 39.6 – 72 x 103 5/5 (100%) 0.22 (± 0.16)
CB CD34- 5 x 106 < 0.01 0/5 < 0.02
a Numbers of CD34+CD38- cells were determined by flow cytometry, using small
aliquots of each preparation of CD34+ enriched cells. These results were obtained in
two very similar, but independent experiments.
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Table 2. Adult human bone marrow contains functional B lineage precursors that
can be enumerated in limiting dilution cultures of CD34+CD38- cells.
Source of CD34+CD38-
cells
Clonogenic Frequency Frequency of cells able to
give rise to CD19+ cells
Cord Blood 12.5a (11,11 – 14,29)b 6.67a (5.56 – 8.33)b
Adult BM 24 years 6.09 (4.84 – 13.53) 1.16 (0.77 – 1.96)
Adult BM 44 years 9.78 (7.78 – 19.01) 1.04 (0.67 – 1.79)
Adult BM 45 years 3.69 (2.78 – 6.56) 0.05 (<0.01 - 0.12)
Adult BM 70 years 4.5 (3.82 – 8.83) 0.18 (0.07 – 0.31)
Adult BM 88 years 3.6 (3.24 – 4.26) 0.15 (0.02 – 0.32)
a Percentage , b95% Confidence Interval (CI).
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Figure Legends
Figure 1. Relative frequencies and mitotic activity of B lineage precursors in
bone marrow do not change during adult life. (A) The incidences of pro B cells
(CD34+CD10+CD19+), pre-B cells (CD19+ sIgM-cµ+), immature B (IgM+CD24hiCD10+IgD-
), naïve B (IgM+CD24hiIgD+CD38+/-) and mature B (IgM+CD24loIgD+CD38-) cells were
determined by flow cytometry and shown as percentages of total viable CD19+
lymphocytes (%) ± S.D. (B) Expression of the Ki-67 nuclear proliferation antigen was
determined by flow cytometry, using low angle light scatter to resolve large and small
lymphocytes. The data represent average percentages of pro B cells (CD34+CD19+),
and pre-B cells (CD19+sIgM-cµ+) ± S.D. Differences between groups were not
statistically significant (p>0,05).
Figure 2. Profiles of immature and naive B cells in some deficient marrow
specimens, reveals a correlation between cycling pre-B cells and immature B
cells. FACS profiles of immature (IgM+CD24hiCD38+IgD-) and naive
(IgM+CD24hiCD38+/-IgD+) B cells from a normal patient (A-B) are compared to a
deficient one (C-D). Immature B cells were also CD10+ (not shown). The index of
cycling pre-B cells in exceptional patient samples correlated with production of
Immature B cells (E), and less closely with naïve B cells (F).
Figure 3. Early lympho-hematopoietic progenitors in adult marrow differ from
ones in cord blood with respect to surface marker characteristics. (A) CD7/CD10
profiles are shown for CD34+ Lin- cells in cord blood and adult marrow. CD34+ Lin-
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CD10- CD7- and CD34+ Lin- CD10- CD7+ cells were sorted from cord blood and adult
marrow, and stained with CD45RA-PE (B) or CD38-PE (C). These data are
representative of three independently sorted samples.
Figure 4. Early lympho-hematopoietic progenitors in cord blood and adult
marrow differ with respect to expansion and differentiation potential. CD34+Lin-
CD10-CD7-, CD34+Lin-CD10-CD7+, and CD34+Lin-CD10+CD7- cells were sorted from
cord blood or adult marrow, and cultured with MS-5 stromal cells (1 x 104 cells/flask in
each fraction). Yields of CD19+ B, CD56+ NK and CD13+/CD33+ myeloid lineage cells
in 3-week cultures are shown (A). The B, NK and myeloid lineage compositions were
also determined weekly in cultures initiated with fractions sorted from cord blood (B) or
adult marrow (C). Similar results were obtained in two independent side-by-side
comparisons between cord blood and adult marrow.
Figure 5. B lineage characteristics are established by the CD10+ stage in adult
marrow. Expression of lineage-related genes was examined in the CD34+Lin-CD10-
CD7- (closed bar), CD34+Lin-CD10-CD7+ (shaded bar) and CD34+Lin-CD10+CD7-
(dotted bar) fractions sorted from adult marrow. RT-PCR was used to amplify
transcripts for the indicated genes and the results were normalized according to
GAPDH expression.
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